World Academy of Science, Engineering and Technology International Journal of Nutrition and Food Engineering Vol:3, No:12, 2009

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1 Manipulation of Probiotics Fermentation of Yogurt by Cinnamon and Licorice: Effects on Yogurt Formation and Inhibition of Helicobacter Pylori Growth in vitro S. Behrad, M.Y. Yusof, K. L. Goh, A.S. Baba Abstract Probiotic bacteria especially Lactobacillus spp. and Bifidobacterium exert suppressive effect on Helicobacter pylori. Cinnamon and licorice have been traditionally used for the treatment of gastric ulcer. The objectives of this study were to determine the effects of herbs on yogurt fermentation, the level of probiotic bacteria in yogurt during 28 days storage and the effect of herbal yogurt on the growth of H. pylori in vitro. Cinnamon or licorice was mixed with milk and the mixture was fermented with probiotic bacteria to form herbal-yogurt. Changes of ph and total titratable acids were monitored and the viability of probiotic bacteria was evaluated during and after refrigerated storage. The in vitro inhibition of H. pylori growth was determined using agar diffusion and minimum inhibitory concentration (MIC) method. The presence of herbs did not affect the probiotic population during storage. There were no significant differences in ph and TTA between herbal-yogurts and plain-yogurt during fermentation and storage. Water extract of cinnamon-yogurt showed the highest inhibition effect (13.5mm) on H. pylori growth in comparison with licorice-yogurt (11.2mm). The present findings indicate cinnamon and licorice has bioactive components to decrease the growth of H. pylori. 1 Keywords Cinnamon, Helicobacter pylori, Herbal-Yogurt, Licorice, Probiotics I. INTRODUCTION ACTIC acid bacteria (LAB) are acid tolerant, Grampositive microorganism, which produce lactic acid as a L main product [1]. The most important genera are Lactobacillus, Lactococcus, Enterocococcus, Streptococcus, Pediococcus, Leuconostoc, and Bifidobacterium. There are several health benefits claimed for probiotic bacteria such as inhibition of Helicobacter pylori and intestinal pathogens, reduction of the risks associated with mutagenicity and carcinogenicity, prevention of inflammatory bowel disease, and improvement of immune system [2]. The suppression of H. pylori growth in vitro by Lactobacillus acidophilus and Bifidobacterium-containing yogurt (AB-yogurt) can be a direct one [3], [4] and as such, consumption of yogurt may S. Behrad is with the Institute of Biological Sciences, University of Malaya, 50603, Kuala Lumpur, Malaysia, phone: ; Fax: ; behrad.sara@yahoo.com M. Y. Yusof is with the Medical Microbiology Department, University of Malaya, 50603, Kuala Lumpur, Malaysia, yasim@um.edu.my K. L. Goh is with department of medicine, faculty of medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia, gohkl@um.edu.my A. S. Baba is with Biochemistry Department, University of Malaya, 50603, Kuala Lumpur, Malaysia, ahmad@um.edu.my exert therapeutic effects by suppressing the growth of H. pylori in infected clinical patients [5], [6]. H. pylori is an important cause of chronic gastritis, peptic ulceration and gastric cancer in humans [7], [8]. It is estimated that one-half of the world's population is infected with H. pylori [9]. Numerous clinical evidences show that eradication of H. pylori results in improvement of gastritis and decreases the rate of relapse of gastric and duodenal ulcers [10], [11]. H. pylori carriage rates are about 80 90% in developing countries [12], with a high risk of gastric cancer and antibiotic resistance [13]. Antibiotics produce undesirable side effects [14] and noncompliance among the patients [15] in the long run. Hence there is a need to develop alternative means to suppress H. pylori infection. In this regard the antibacterial activity of several plant extracts have been tested in vitro [17], a number of which were effective against H. pylori growth. In addition, yogurt bacteria were reported effective in suppressing H. pylori infection [18]. The present study investigated the effects of herbs on yogurt fermentation and viability of yogurt bacteria during refrigerated storage, and the effect of yogurts on H. pylori growth in vitro. II. A. Preparation of extracts MATERIALS AND METHODS Stem barks of cinnamon and roots of licorice were obtained from a local Chinese medicinal shop. Both were ground to fine powder. The powdered herbs (10g) were soaked in 100ml of distilled water and left overnight at 70 C. The suspension was then centrifuged (2000 rpm; 15 min), and the supernatant was sterilized through 0.22 μm filter (Sartorius, Germany). B. Herbal yogurt preparation Homogenized and pasteurized milk was purchased from the local supermarket. Starter culture (5g) consisting of L. acidophilus LA-5 and NCFM, Bifidobacterium Bb-12, L.casei LC-10, and Streptococcus thermophilus Th-4, 2% (w/v) skim milk powder and 6% (w/v) herb extract were dissolved in 1L of milk. The mixtures were aliquoted in 100ml plastic cups. Incubation was carried out at 41 C, and fermentation was terminated at ph 4.5. C. Determination of ph and TTA 563

2 The ph and TTA of yogurts were determined every hour at C during fermentation and storage at 4 C. Yogurt sample (1g) was mixed with distilled water (1:1), and the ph was measured using a ph meter (Mettler-Toledo 320, Shanghai), calibrated routinely with fresh ph 4.0 and 7.0 standard buffers. TTA was determined by titrating yogurt sample and distilled water (1:9) mixture with 0.1N NaoH using a 0.1% Phenolphetalein as color indicator. The amount of acid produced during fermentation was calculated as follows: TTA% = Dilution factor (10) x V NaoH x 0.1N x x 100% where V is volume of NaoH required to neutralize the acid. D. Enumeration of probiotic bacteria Enumeration of Lactobacillus spp was carried out by aseptically mixing yogurt sample (1ml) with 9ml of buffered peptone water (Oxoid,UK). The sample was thoroughly mixed and serial dilutions were performed using peptone water as the diluents. Empty petri dishes were inoculated with 1ml of diluted yogurt, followed by the addition of 15ml melted (45 C) MRS agar. The petri dishes were covered and the contents mixed thoroughly by gentle tilting and swirling. The petri dishes were inverted and incubated anaerobically (Revco Ultima) at 37 C for hours. Streptococci was enumerated by initially placing 15ml of melted (45 C) M17 (Oxoid, UK) into a petri dish followed by cooling of agar to temperature to allow solidification. The agar was then inoculated by spreading the surface evenly with 0.1ml of diluted yogurt. The colonies formed were counted after hour incubation at 37 C. Viable microbial count was calculated as follows: cfu/ml = cfu/plate x dilution factor where cfu is colony forming unit E. Bacterial isolates Clinical isolates of H. pylori were obtained from the University of Malaya Medical Center, Kuala Lumpur, Malaysia. The organisms were identified based on colony morphology, Gram staining, microaerophilic growth (at 37 C), oxidase, catalase, and urease assays. In the present study, two clinical isolates (numbered as UM-1 and UM-2) from hospital patients were used. The growth of H. pylori was maintained under microaerophilic conditions in anaerobic jars with CampyPakPlus (MGC Anaeropack, Microaero) at 37 C for 3 5 days. Bacterial strains were suspended in brain heart infusion broth (BHIB) (Oxoid, UK) containing 15% (v/v) glycerol and stored at -70 C. F. Bacterial growth inhibition assay Growth inhibition was evaluated by the filter paper disk diffusion method [21] which conforms to the recommended standards of National Committee for Clinical Laboratory Standards (NCCLS). Each of herbal-yogurt water extracts (25 μl) was aliquoted on standard 6 mm paper disks (Whatman, UK) which were then placed on Columbia agar supplemented with 7% sheep blood (BML, Malaysia), and inoculated with 0.1 ml bacterial suspension ( cfu/ml) in the brain heart infusion broth (BHIB). The growth of H. pylori was maintained under microaerophilic conditions in anaerobic jars with gas pack (to absorb oxygen and generating carbon dioxide) at 37 C for 3-5 days. The inhibition zone around each disk (average of triplicate) was measured. The minimal inhibition concentration (MIC) was determined by mixing various volumes ( ml) of herbalyogurt water extract with heated (50 C) Mueller Hinton blood agar, before inoculation with H. pylori suspension [22]. G. Antioxidant activity by 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) To 3ml of 60μM DPPH in ethanol, 250μl of each herbalyogurt water extracts was added and the decrease in absorbance was measured at 517 nm. The readings were compared with the controls, which contained 250μl of dh 2 O instead of the water extracts. The % of antioxidant activity inhibition was calculated as follows: A control-a extract A control %inhibition= x 100 III. 517 RESULTS AND DISCUSSION A. ph changes in yogurts during storage The ph for plain yogurt was approximately the same as ph of herbal yogurts. An overall decline of ph of yogurts occurred during refrigerated storage. The ph for all yogurts reduced (p<0.05) from the initial values of 4.5 to between 4.09 and 4.12 by day 28 of storage. The presence of herbs did not make herbal-yogurts any different (p>0.05) from plainyogurt. Fig. 1 Changes in ph of plain, cinnamon- and licorice-yogurts during refrigerated (4 C) storage. Cinnamon ( ); Licorice ( ); Plain ( ) 564

3 Fig. 2 Changes of total titratable acid (TTA) of plain, cinnamon- and licorice-yogurts during refrigerated (4 C) storage. Cinnamon ( ); Licorice ( ); Plain ( ) B. Changes in Total titraable acid of yogurts during storage The TTA of all yogurts increased (p<0.05) from the initial values of 1% to 1.27% by day 28 of storage. The increase in acids can be attributed to continued production of organic acids by LAB during refrigerated storage [20]. C. Survival of probiotic bacteria The presence of cinnamon or licorice resulted in lower Lactobacillus spp counts in cinnamon- (9.46 x 10 6 cfu/ml) and licorice- (12.3 x 10 6 cfu/ml) yogurts on 0 day of storage compared to plain-yogurt (12.96 x 10 6 cfu/ml; p>0.05). Refrigeration increased (p>0.05) viable Lactobacillus spp. counts to 15.8 x 10 6 cfu/ml in the plain-yogurt but the presence of cinnamon or licorice inhibited this increase in herbalyogurts. Viable Lactobacillus spp counts reduced from day 7 to day 28 of storage for all yogurts with the fastest rate occurred in plain-yogurt. Viable Lactobacillus spp counts on day 28 of storage for licorice-yogurt (6.4 x 10 6 cfu/ml) was higher than plain-yogurt (4 x 10 6 cfu/ml). Refrigeration also increased (p>0.05) viable S. thermophillus counts in all yogurts by day 7 of refrigeration (Fig.4) but the effect was significant (p<0.05) only in the presence of cinnamon and licorice (140 x 10 6 cfu/ml) compared to plain-yogurt (115 x 10 6 cfu/ml). The increase in the viable cell counts for both yogurt bacteria during the first 7 days coincided with the increase in TTA and marked reduction (p<0.05) in ph recorded on day 7 of storage. The reduction in viable cell counts which occurred in consistent manner in all yogurts can be attributed to the organic acids accumulation as a result of growth and fermentation (Figs. 1 & 2), [19], and [20]. Nevertheless, all yogurts contained acceptable level of probiotic bacteria ( cfu/ml) by the end of 28 day of refrigerated storage. D. DPPH inhibition assay The addition of cinnamon or licorice increased the antioxidant activity of yogurts compared to plain-yogurt at all storage periods (Fig. 5). The highest antioxidant activity was recorded on day 7 for cinnamon-yogurt (31.8%) followed by licorice-yogurt (23.87%) and plain-yogurt (21.8%). Cinnamon-yogurt showed the highest antioxidant activity on day 0 (p>0.05) and 14 (p<0.05) of storage. Fig. 3 Effect Viable Lactobacillus spp.in cinnamon- or licorice-yogurts during refrigerated storage. 565

4 Fig. 4 Viable S. thermophilus in cinnamon- or licorice-yogurts during refrigerated storage. E. Bacterial inhibition assay Fig. 5 DPPH radical inhibition capacity of water extracts from plain and herbal-yogurts Water extract of cinnamon-yogurt exhibited the strongest inhibitory effect on H. pylori growth in vitro (13.5mm) in comparison with licorice-yogurt (11.2mm) and control-yogurt (10.5mm) for both strains tested. Licorice-yogurt extract at volume of 1ml had an inhibitory effect on H. pylori growth for both strain UM-1 and UM-2. However cinnamon-yogurt can only inhibit H. pylori growth at a volume of 3 and 2 ml for UM-1 and UM-2 strains respectively. It can be concluded that the addition of cinnamon or licorice did not change yogurt fermentation but sustain the growth of Lactobacillus spp during refrigerated storage. Cinnamon-yogurt or licorice-yogurt containing probiotic bacteria inhibited the growth of H. pylori in vitro. The effectiveness of these herbal-yogurts to halt the growth of H. pylori needs to be further investigated under extremely acidic environment of the stomach. TABLE I GROWTH INHIBITION OF H. PYLORI BY YOGURT WATER EXTRACT Inhibition Zone (mm) Herbal-Yogurt Water Extract (mg/ml) Isolate Number Cinnamo n-yogurt Licorice -yogurt Controlyogurt UM UM

5 TABLE II MINIMUM INHIBITORY CONCENTRATION (MIC) OF YOGURT WATER EXTRACTS ON THE GROWTH OF H. PYLORI ISOLATES a Isola tes num ber 1 2 Antimicrobial agent Volume of yogurt or herbal yogurt water extract (ml) 0 (blank) Cinnamonyogurt Licorice-yogurt _ UM- UM- Plain-yogurt Cinnamonyogurt Licorice-Yogurt Plain-yogurt a Note: the following notations estimated H. pylori growth as follows:, no growth; +, scant growth; + +, moderate growth; + + +, extensive growth; , very extensive growth. ACKNOWLEDGMENT The authors thank Ms. Chan Chui Lin for her assistance in the isolation, growing and storing H. pylori isolates. REFERENCES [1] Stiles, M. E., & Holzapfel, W. H. Lactic acid bacteria of foods and their current taxonomy. International Journal of Food Microbiology, 1997, 36: [2] Metchnikoff, I. I. The prolongation of life: Optimistic studies (reprinted edition 1907). New York, NY, USA: Springer [3] Coconnier MH, Lievin V, Hemery E. Antagonistic activity against Helicobacter infection in vitro and in vivo by the human Lactobacillus acidophilus strain LB. Appl Environ Microbiol, 1998, 64: [4] Midolo PD, Lambert JR, Hull R. In vitro inhibition of Helicobacter pylori NCTC by organic acids and lactic acid bacteria. J Appl Bacteriol, 1995, 79: [5] Sheu BS, Wu JJ, Lo CY. Lactobacillus and Bifidobacteria containing yogurt can improve the drug compliance of triple therapy for Helicobacter pylori eradication. Aliment Pharmacol Ther, 2002, 16: [6] Wang KY, Li SN, Liu C. Effects of ingesting Lactobacillus- and Bifidobacterium-containing yogurt in subjects with colonized Helicobacter pylori. Am J Clin Nutr, 2004, 80: [7] Borsch G. Campylobacter pylori: new and renewed insights into gastritis-associated ulcer disease (GAUD). Hepato-Gastroenterology, 1987, 34: [8] Tytgat GN, Rauws EA. The role of Campylobacter pylori in gastroduodenal diseases. A believer s point of view. Gastroenterol Clin Bio, 1989, 13:118B 21B. [9] EUROGAST study group: An International Association between Helicobacter pylori infection and Gastric Cancer. Lancet 1993, 341: [10] Marshall BJ, Goodwin CS, Warren JR, Murray R, Blincow ED, Blackbourn SJ, Phillips M, Waters TE, and Sanderson CR: Prospective double blind trail of duodenal ulcer relapse after eradication of Campylobacter pylori. Lancet 1988, 2: [11] Rauws EA, Langenberg W, Houthoff HJ, Zanen HC, Tytgat GN: Campylobacter pyloridis -associated chronic active antral gastritis. A prospective study of its prevalence and the effects of antibacterial and antiulcer treatment. Gastroenterol 1988, 94: [12] Lacy BE, Rosemore J: Helicobacter pylori: ulcers and more: the beginning of an era. J Nutr 2001, 131: 2789S-2793S [13] Buiatti E, Munoz N, Vivas J, Cano E, Peraza S: Difficulty in eradicating Helicobacter pylori in a population at high risk for gastric cancer in Venezuela. Cancer Causes Control 1994, 5: [14] Bell GD, Powell KU, Burridge SM, Bowden AN, Ramesh B, Bolten G, Purser K, Harrison G, Brown C, Gant PW, Jones PH, Trowell JE: Helicobacter pylori eradication: efficacy and side effect profile of a combination of omeprazole, amoxycillin and metronidazole compared with four alternative regimens. QJ Med J Assoc Phys 1993, 86: [15] Broutet N, Tchamgoue S, Pereira E, Lamouliatte H, Salamon R, Megraud F: Risk factors for failure of Helicobacter pylori therapyresults of an individual data analysis of 2751 patients. Aliment Pharmacol Ther 2003, 17: [16] O'Gara EA, Hill DJ, Maslin DJ: Activities of garlic oil, garlic powder, and their diallyl constituents against Helicobacter pylori. Appl Environ Microbiol 2000, 66: [17] Tabak M, Armon R, Potasman I, Neeman I. In vitro inhibition of Helicobacter pylori by extracts of Thyme. J Appl Bacteriol 1996, 80: [18] Sheu B-S, Wu J-J, Lo C-Y, et al. Impact of supplement with Lactobacillus - and Bifidobacterium-containing yogurt on triple therapy for Helicobacter pylori eradication. Aliment Pharmacol Ther 2002, 16: [19] Hood, S. K., & Zottola, M. L. Effects of low PH on the ability of Lactobacillus acidophilus to survive and adhere to human intestinal cells. J Food Science 1998, 53: [20] Shah, N. P. Probiotic bacteria, Selective enumeration and survival in dairy foods. Journal of Dairy Science 2000, 83: [21] Zaika, L.L. (1988). Spices and herbs: their antimicrobial activity and its determination. J Food Safety 1988, 9: [22] Shahamat, M., Mai, U.E.H., Paszko-Kolva, C., Yamamoto, H., Colwell, R.R. Evaluation of liquid media for growth of Helicobacter pylori. J. Clin. Microbiol 1991, 29: